Thioflavin S-positive Plaques Research Articles

NMDA receptor antagonists reduce amyloid-β deposition by modulating calpain-1 signaling and autophagy, rescuing cognitive impairment in 5XFAD mice

Overstimulation of N-methyl-d-aspartate receptors (NMDARs) is the leading cause of brain excitotoxicity and often contributes to neurodegenerative diseases such as Alzheimer’s Disease (AD), the most common form of dementia. This study aimed to evaluate a new NMDA receptor antagonist (UB-ALT-EV) and memantine in 6-month-old female 5XFAD mice that were exposed orally to a chronic low-dose treatment. Behavioral and cognitive tests confirmed better cognitive performance in both treated groups. Calcium-dependent protein calpain-1 reduction was found after UB-ALT-EV treatment but not after memantine. Changes in spectrin breakdown products (SBDP) and the p25/p35 ratio confirmed diminished calpain-1 activity. Amyloid β (Aβ) production and deposition was evaluated in 5XFAD mice and demonstrated a robust effect of NMDAR antagonists on reducing Aβ deposition and the number and size of Thioflavin-S positive plaques. Furthermore, glycogen synthase kinase 3β (GSK3β) active form and phosphorylated tau (AT8) levels were diminished after UB-ALT-EV treatment, revealing tau pathology improvement. Because calpain-1 is involved in autophagy activation, autophagic proteins were studied. Strikingly, results showed changes in the protein levels of unc-51-like kinase (ULK-1), beclin-1, microtubule-associated protein 1A/1B-light chain 3(LC3B-II)/LC3B-I ratio, and lysosomal-associated membrane protein 1 (LAMP-1) after NMDAR antagonist treatments, suggesting an accumulation of autophagolysosomes in 5XFAD mice, reversed by UB-ALT-EV. Likewise, treatment with UB-ALT-EV recovered a WT mice profile in apoptosis markers Bcl-2, Bax, and caspase-3. In conclusion, our results revealed the potential neuroprotective effect of UB-ALT-EV by attenuating NMDA-mediated apoptosis and reducing Aβ deposition and deposition jointly with the autophagy rescue to finally reduce cognitive alterations in a mice model of familial AD.

Selective drug inhibition of rho kinase II (ROCK2) reduces beta-amyloid production in the brain

worse than apoE4-TR mice. Ab12-28P treatment of APP SW/PS1 dE9/ apoE4-TR mice normalized OR behavior and improved RAM performance to the level of apoE4-TR mice. Levels of soluble and insoluble Ab x-40 and Ab x-42 measured by ELISA in the whole brain extract and the burden of Thioflavin-S positive plaques were significantly higher in APP SW/PS1 dE9/apoE4-TR than in APP SW/PS1 dE9/apoE2-TR mice. Ab12-28P treatment was associated with significant reduction in soluble and insoluble Ab x-40 and Ab x-42 levels and lower Thioflavin-S plaque burden in the neocortex and in the hippocampus in both APP SW/PS1 dE9/apoE2-TR and APP SW/PS1 dE9/apoE4-TR lines. Conclusions: Our results indicate that future therapies targeting the apoE/Ab interaction could produce favorable outcome in both APOE2 and APOE4 carriers.

Deletion ofAbca7Increases Cerebral Amyloid-β Accumulation in the J20 Mouse Model of Alzheimer's Disease

ATP-binding cassette transporter A7 (ABCA7) is expressed in the brain and has been detected in macrophages, microglia, and neurons. ABCA7 promotes efflux of lipids from cells to apolipoproteins and can also regulate phagocytosis and modulate processing of amyloid precursor protein (APP) to generate the Alzheimer's disease (AD) amyloid-β (Aβ) peptide. Genome-wide association studies have indicated that ABCA7 single nucleotide polymorphisms confer increased risk for late-onset AD; however, the role that ABCA7 plays in the brain in the AD context is unknown. In the present study, we crossed ABCA7-deficient (A7(-/-)) mice with J20 amyloidogenic mice to address this issue. We show that ABCA7 loss doubled insoluble Aβ levels and thioflavine-S-positive plaques in the brain. This was not related to changes in APP processing (assessed by analysis of full-length APP and the APP β C-terminal fragment). Apolipoprotein E regulates cerebral Aβ homeostasis and plaque load; however, the apolipoprotein E concentration was not altered by ABCA7 loss. Spatial reference memory was significantly impaired in both J20 and J20/A7(-/-) mice compared with wild-type mice; however, there were no cognitive differences between J20 and J20/A7(-/-) mice. There were also no major differences detected in hippocampal or plaque-associated microglial/macrophage markers between J20 and J20/A7(-/-) mice, whereas the capacity for bone marrow-derived macrophages derived from A7(-/-) mice to take up oligomeric Aβ was reduced by 51% compared with wild-type mice. Our results suggest that ABCA7 plays a role in the regulation of Aβ homeostasis in the brain and that this may be related to altered phagocyte function.

P1‐279: APP knock‐in mouse: A novel and more relevant model mouse for Alzheimer's disease

Mouse models of Alzheimer's disease (AD) exhibiting amyloidgenic pathology are most useful tools for AD research and play important roles in the development of new therapeutic approaches and treatments. However, APP transgenic mice over express APP and even though FAD mutations ensures high production of Aß species the by-products sAPP and intracellular domain of APP (AICD) are also generated, both of which have physiological functions. This leads to uncertainties as to whether the various observed phenotypes are caused by increased Aß or by the overproduction of sAPP, AICD or APP. To address these issues, we generated a novel type of AD mouse model by applying a gene knock-in strategy. We constructed a targeting vector of the mouse APP gene that included the humanized Aß sequence harboring the Swedish mutation (KM670/671NL) and the Iberian mutation (I716F). The knock-in mice were investigated by biochemical, pathological and behavioral studies. The mutations in the generated mice (APP-NL/F-KI), gave rise to increased Aß 42 and decreased Aß 40 levels even in heterozygous mice (wt/NL-F) and already at the age of 2 month, so that Aß 42/A b 40 ratios were >15 fold increased in the brain compared to wild type mice. In addition, A b 42/A b 40 ratios of the homozygous (NL-F/NL-F) were >50 fold increased vs. wild type. So far, depositions of Aß 1-42, in partially thioflavin-S positive plaques, were visible from 6 month age of NL-F/NL-F mice. 5 months old homozygous mice also showed behavioral abnormality. In 12 month old NL-F/NL-F mice brain, accumulation of N3pE-Aß species and Aßx- 43 species could be detected. In addition, tau phosphorylation was also accelerated in 12 month old NL-F/NL-F mice. We are currently assessing this mouse model at older ages with respect to different paradigms. Such mice, after crossbreeding with other type of mouse models for AD, would be powerful mouse models in AD research.

P1‐280: Behavioural and cerebral metabolism assessment of TASTPM transgenic mice using a ‘touchscreen’‐based cognitive task

However, APP transgenic mice over express APP and even though FADmutations ensures high production of As species the by-products sAPP and intracellular domain of APP (AICD) are also generated, both of which have physiological functions. This leads to uncertainties as to whether the various observed phenotypes are caused by increased As or by the overproduction of sAPP, AICD or APP. To address these issues, we generated a novel type of AD mouse model by applying a gene knock-in strategy.Methods:We constructed a targeting vector of the mouse APP gene that included the humanized As sequence harboring the Swedish mutation (KM670/671NL) and the Iberian mutation (I716F). The knock-in micewere investigated by biochemical, pathological and behavioral studies.Results: The mutations in the generated mice (APP-NL/F-KI), gave rise to increased As 42 and decreased As 40 levels even in heterozygous mice (wt/NL-F) and already at the age of 2 month, so that As 42/A b 40 ratios were >15 fold increased in the brain compared to wild type mice. In addition, A b 42/A b 40 ratios of the homozygous (NL-F/NL-F) were>50 fold increased vs. wild type. So far, depositions of As 1-42, in partially thioflavin-S positive plaques, were visible from 6 month age of NL-F/NL-F mice. 5 months old homozygous mice also showed behavioral abnormality. In 12month old NL-F/NL-Fmice brain, accumulation of N3pE-As species and Asx43 species could be detected. In addition, tau phosphorylation was also accelerated in 12 month old NL-F/ NL-F mice. Conclusions: We are currently assessing this mouse model at older ages with respect to different paradigms. Such mice, after crossbreeding with other type of mouse models for AD, would be powerful mouse models in AD research.

Deletion of CD14 Attenuates Alzheimer's Disease Pathology by Influencing the Brain's Inflammatory Milieu

Alzheimer's disease (AD) is characterized by the deposition of β-amyloid (Aβ)-containing plaques within the brain that is accompanied by a robust microglial-mediated inflammatory response. This inflammatory response is reliant upon engagement of innate immune signaling pathways involving the toll-like receptors (TLRs). Studies assessing the roles of TLRs in AD pathogenesis have yielded conflicting results. We have assessed the roles of the TLRs through genetic inactivation of the TLR2/4 coreceptor, CD14, in a transgenic murine model of AD. Transgenic mice lacking CD14 exhibited reduced insoluble, but not soluble, levels of Aβ at 7 months of age. This corresponded with decreased plaque burden resulting from a reduction in number and size of both diffuse and thioflavin S-positive plaques and an overall reduction in the number of microglia. These findings are inconsistent with the established actions of these receptors. Moreover, loss of CD14 expression was associated with increased expression of genes encoding the proinflammatory cytokines Tnfα and Ifnγ, decreased levels of the microglial/macrophage alternative activation markers Fizz1 and Ym1, and increased expression of the anti-inflammatory gene Il-10. Thus, the loss of CD14 resulted in a significant change in the inflammatory environment of the brain, likely reflecting a more heterogeneous population of microglia within the brains of the animals. The reduction in plaque burden was not a result of changes in the expression of various Aβ degrading enzymes or proteins associated with Aβ clearance. These data suggest that CD14 is a critical regulator of the microglial inflammatory response that acts to modulate Aβ deposition.

Tyrosine phosphorylation of tau accompanies disease progression in transgenic mouse models of tauopathy

Tau protein is a prominent component of paired helical filaments in Alzheimer's disease (AD) and other tauopathies. While the abnormal phosphorylation of tau on serine and threonine has been well established in the disease process, its phosphorylation on tyrosine has only recently been described. We previously showed that the Src family non-receptor tyrosine kinases (SFKs) Fyn and Src phosphorylate tau on Tyr18 and that phospho-Tyr18-tau was present in AD brain. In this study, we have investigated the appearance of phospho-Tyr18-tau, activated SFK and proliferating cell nuclear antigen (PCNA) during disease progression in a mouse model of human tauopathy. We have used JNPL3, which expresses human tau with P301L mutation, and antibodies specific for phospho-Tyr18-tau (9G3), ser/thr phosphorylated tau (AT8), activated SFK and PCNA. Antibody staining was viewed by either epifluorescence or confocal microscopy. Phospho-Tyr18-tau appeared concurrently with AT8-reactive tau as early as 4 months in JNPL3. Some 9G3-positive cells also contained activated SFKs and PCNA. We also investigated the triple transgenic mouse model of AD and found that unlike the JNPL3 model, the appearance of 9G3 reactivity did not coincide with AT8 in the hippocampus, suggesting that the presence of APP/presenilin influences tau phosphorylation. Also, Thioflavin S-positive plaques were 9G3-negative, suggesting that phospho-Tyr18-tau is absent from the dystrophic neurites of the mouse triple transgenic brain. Our results provide evidence for the association of tyrosine-phosphorylated tau with mechanisms of neuropathogenesis and indicate that SFK activation and cell cycle activation are also involved in JNPL3.

β-Amyloid Deposition and Functional Impairment in the Retina of the APPswe/PS1ΔE9 Transgenic Mouse Model of Alzheimer’s Disease

To determine whether beta-amyloid (Abeta) deposition affects the structure and function of the retina of the APPswe/PS1DeltaE9 transgenic (tg) mouse model of Alzheimer's disease. Retinas from 12- to 19-month old APPswe/PS1DeltaE9 tg and age-matched non-transgenic (ntg) littermates were single or double stained with thioflavine-S and antibodies against Abeta, glial fibrillar acidic protein (GFAP), microglial marker F4/80, choline acetyltransferase (ChAT), and syntaxin 1. Quantification of thioflavine-S positive plaques and retinal layer thickness was analyzed semi-quantitatively, whereas microglial cell size and levels of F4/80 immunoreactivity were evaluated using a densitometry program. Scotopic electroretinogram (ERG) recording was used to investigate retinal physiology in these mice. Thioflavine-S positive plaques appeared at 12 months in the retinas of APPswe/PS1DeltaE9 tg mice with the majority of plaques in the outer and inner plexiform layers. Plaques were embedded in the inner plexiform layer strata displaying syntaxin 1 and ChAT. The number and size of the plaques in the retina increased with age. Plaques appeared earlier and in greater numbers in females than in male tg littermate mice. Microglial activity was significantly increased in the retinas of APPswe/PS1DeltaE9 tg mice. Although we did not detect neuronal degeneration in the retina, ERG recordings revealed a significant reduction in the amplitudes of a- and b-waves in aged APPswe/PS1DeltaE9 tg compared to ntg littermates. The present findings suggest that Abeta deposition disrupts retinal structure and may contribute to the visual deficits seen in aged APPswe/PS1DeltaE9 tg mice. Whether Abeta is involved in other forms of age-related retinal dysfunction is unclear.

Antiamyloidogenic and Neuroprotective Functions of Cathepsin B: Implications for Alzheimer's Disease

Alzheimer's disease (AD) may result from the accumulation of amyloid-beta (Abeta) peptides in the brain. The cysteine protease cathepsin B (CatB) is associated with amyloid plaques in AD brains and has been suspected to increase Abeta production. Here, we demonstrate that CatB actually reduces levels of Abeta peptides, especially the aggregation-prone species Abeta1-42, through proteolytic cleavage. Genetic inactivation of CatB in mice with neuronal expression of familial AD-mutant human amyloid precursor protein (hAPP) increased the relative abundance of Abeta1-42, worsening plaque deposition and other AD-related pathologies. Lentivirus-mediated expression of CatB in aged hAPP mice reduced preexisting amyloid deposits, even thioflavin S-positive plaques. Under cell-free conditions, CatB effectively cleaved Abeta1-42, generating C-terminally truncated Abeta peptides that are less amyloidogenic. Thus, CatB likely fulfills antiamyloidogenic and neuroprotective functions. Insufficient CatB activity might promote AD; increasing CatB activity could counteract the neuropathology of this disease.

Lack of ABCA1 Considerably Decreases Brain ApoE Level and Increases Amyloid Deposition in APP23 Mice

ABCA1 (ATP-binding cassette transporter A1) is a major regulator of cholesterol efflux and high density lipoprotein (HDL) metabolism. Mutations in human ABCA1 cause severe HDL deficiencies characterized by the virtual absence of apoA-I and HDL and prevalent atherosclerosis. Recently, it has been reported that the lack of ABCA1 causes a significant reduction of apoE protein level in the brain of ABCA1 knock-out (ABCA1-/-) mice. ApoE isoforms strongly affect Alzheimer disease (AD) pathology and risk. To determine further the effect of ABCA1 on amyloid deposition, we used APP23 transgenic mice in which the human familial Swedish AD mutant is expressed only in neurons. We demonstrated that the targeted disruption of ABCA1 increases amyloid deposition in APP23 mice, and the effect is manifested by an increased level of Abeta immunoreactivity, as well as thioflavine S-positive plaques in brain parenchyma. We found that the lack of ABCA1 also considerably increased the level of cerebral amyloid angiopathy and exacerbated cerebral amyloid angiopathy-related microhemorrhage in APP23/ABCA1-/- mice. Remarkably, the elevation in parenchymal and vascular amyloid in APP23/ABCA1-/- mice was accompanied by a dramatic decrease in the level of soluble brain apoE, although insoluble apoE was not changed. The elevation of insoluble Abeta fraction in old APP23/ABCA1-/- mice, accompanied by a lack of changes in APP processing and soluble beta-amyloid in young APP23/ABCA1-/- animals, supports the conclusion that the ABCA1 deficiency increases amyloid deposition. These results suggest that ABCA1 plays a role in the pathogenesis of parenchymal and cerebrovascular amyloid pathology and thus may be considered a therapeutic target in AD.

Growth Arrest of Individual Senile Plaques in a Model of Alzheimer's Disease Observed byIn VivoMultiphoton Microscopy

In Alzheimer's disease, amyloid-beta peptide aggregates in the extracellular space to form senile plaques. The process of plaque deposition and growth has been modeled on the basis of in vitro experiments in ways that lead to divergent predictions: either a diffusion-limited growth model in which plaques grow by first-order kinetics, or a dynamic model of continual deposition and asymmetrical clearance in which plaques reach a stable size and stop growing but evolve morphologically over time. The models have not been tested in vivo because plaques are too small (by several orders of magnitude) for conventional imaging modalities. We now report in vivo multiphoton laser scanning imaging of thioflavine S-stained senile plaques in the Tg2576 transgenic mouse model of Alzheimer's disease to test these biophysical models and show that there is no detectable change in plaque size over extended periods of time. Qualitatively, geometric features remain unchanged over time in the vast majority of the 349 plaques imaged and re-imaged. Intervals as long as 5 months were obtained. Nonetheless, rare examples of growth or shrinkage of individual plaques do occur, and new plaques appear between imaging sessions. These results indicate that thioflavine S-positive plaques appear and then are stable, supporting a dynamic feedback model of plaque growth.

Acetylcholinesterase-amyloid-beta-peptide interaction and Wnt signaling involvement in Abeta neurotoxicity.

Previous studies have indicated that acetylcholinesterase (AChE) promotes amyloid-beta-peptide (Abeta) fibril formation and AChE-Abeta complexes increase Abeta-dependent neurotoxicity. Here we present evidence for the: i) identification of the AChE motif that promotes amyloid formation, ii) in vivo effect of AChE on brain plaque formation, and iii) connection between AChE-Abeta neurotoxicity and the Wnt signal transduction pathway. Computer modeling, stereotaxic infusions and cell biological techniques were used to study the above problems. Results indicated that a 3.4 kDa AChE peptide promotes Abeta fibril formation. AChE infusion into rat hippocampus determines the appearance of anti-Abeta and thioflavine-S positive plaques, and AChE-Abeta toxicity on hippocampal cultures was blocked by lithium, an activator of the Wnt cascade. We suggest that AChE-Abeta/Abeta dependent neurotoxicity may result in loss of function of Wnt signaling components, and open the possibility that lithium may be considered as a candidate for therapeutic intervention in Alzheimer's disease pathology.